Inflammation is a response of vascularized tissues to infection or injury and is effected by adhesion of leukocytes to the endothelial cells of blood vessels and their infiltration into the surrounding tissues. In normal inflammation, the infiltrating leukocytes release toxic mediators to kill invading organisms, phagocytize debris and dead cells, and play a role in tissue repair and the immune response. However, in pathologic inflammation, infiltrating leukocytes are over-responsive and can cause serious or fatal damage. See, e.g., Hickey, Psychoneuroimmunology II (Academic Press 1990).
The attachment of leukocytes to endothelial cells is effected via specific interaction of cell-surface ligands and receptors on endothelial cells and leukocytes. See generally Springer, Nature 346:425-433 (1990). The identity of the ligands and receptors varies for different cell subtypes, anatomical locations and inflammatory stimuli. The VLA-4 leukocyte cell-surface receptor was first identified by Hemler, EP 330,506 (1989) (incorporated by reference in its entirety for all purposes). VLA-4 is a member of the β1 integrin family of cell surface receptors, each of which comprises α and β chains. VLA-4 contains an α4 chain and a β1 chain. VLA-4 specifically binds to an endothelial cell ligand termed VCAM-1. See Elices et al., Cell 60:577-584 (1990) (incorporated by reference in its entirety for all purposes). The α4 chain also associates with a β7 chain to form an integrin referred to as α4β7. Although VCAM-1 was first detected on activated human umbilical vein cells, this ligand has also been detected on brain endothelial cells. See commonly owned, co-pending application U.S. Ser. No. 07/871,223 (incorporated by reference in its entirety for all purposes).
Adhesion molecules such as α4 integrin are potential targets for therapeutic agents. The VLA-4 receptor of which α4 integrin is a subunit is a particularly important target because of its interaction with a ligand residing on brain endothelial cells. Diseases and conditions resulting from brain inflammation have particularly severe consequences. For example, one such disease, multiple sclerosis (MS), has a chronic course (with or without exacerbations and remissions) leading to severe disability and death. The disease affects an estimated 250,000 to 350,000 people in the United States alone.
Antibodies against α4 integrin have been tested for their anti-inflammatory potential both in vitro and in vivo in animal models. See U.S. Ser. No. 07/871,223 and Yednock et al., Nature 356:63-66 (1992) (incorporated by reference in its entirety for all purposes). The in vitro experiments demonstrate that α4 integrin antibodies block attachment of lymphocytes to brain endothelial cells. The animal experiments test the effect of α4 integrin antibodies on animals having an artificially induced condition (experimental autoimmune encephalomyelitis), simulating multiple sclerosis. The experiments show that administration of anti-α4 integrin antibodies prevents inflammation of the brain and subsequent paralysis in the animals. Collectively, these experiments identify anti-α4 integrin antibodies as potentially useful therapeutic agents for treating multiple sclerosis and other inflammatory diseases and disorders.
A significant problem with the anti-α4 integrin antibodies available to-date is that they are all of murine origin, and therefore likely to raise a human anti-mouse response (HAMA) in clinical use. A HAMA response reduces the efficacy of mouse antibodies in patients and prevents continued administration. One approach to this problem is to humanize mouse antibodies. In this approach, complementarity determining regions (CDRs) and certain other amino acids from donor mouse variable regions are grafted into human variable acceptor regions and then joined to human constant regions. See, e.g., Riechmann et al., Nature 332:323-327 (1988); Winter, U.S. Pat. No. 5,225,539 (1993) (each of which is incorporated by reference in its entirety for all purposes).
Although several examples of humanized antibodies have been produced, the transition from a murine to a humanized antibody involves a compromise of competing considerations, the solution of which varies with different antibodies. To minimize immunogenicity, the immunoglobulin should retain as much of the human acceptor sequence as possible. However, to retain authentic binding properties, the immunoglobulin framework should contain sufficient substitutions of the human acceptor sequence to ensure a three-dimensional conformation of CDR regions as close as possible to that in the original mouse donor immunoglobulin. As a result of these competing considerations, many humanized antibodies produced to-date show some loss of binding affinity compared with the corresponding murine antibodies from which they are derived. See, e.g., Jones et al., Nature 321:522-525 (1986); Shearman et al., J. Immunol. 147:4366-4373 (1991); Kettleborough et al., Protein Engineering 4:773-783 (1991); Gorman et al., Proc. Natl. Acad. Sci. USA 88:4181-4185 (1991); Tempest et al., Biotechnology 9:266-271 (1991).
Based on the foregoing it is apparent that a need exists for humanized anti-α4 integrin antibodies demonstrating a strong affinity for α4 integrin, while exhibiting little, if any, human-antimouse response. The present invention fulfill this and other needs.